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Manlio Ferrarini

Publications and source records attributed to Manlio Ferrarini.

At least 19 recordsLinked to original sources

To Treat or Not to Treat: Navigating Early-Stage CLL in the Era of Targeted Therapy.

Chronic lymphocytic leukemia (CLL) is most frequently diagnosed at early, asymptomatic stages (Rai 0/Binet A), in which a watch-and-wait strategy remains the standard of care, based on historical trials demonstrating no overall survival benefit from early treatment. Over the past two decades, however, substantial advances in genomic profiling-including immunoglobulin heavy-chain variable region (IGHV) mutational status, TP53 disruption, recurrent gene mutations, and complex karyotype-have uncovered marked biological heterogeneity among early-stage patients and substantially improved prediction of disease progression. In parallel, targeted therapies such as Bruton tyrosine kinase (BTK) inhibitors and venetoclax-based combinations have transformed the management of symptomatic CLL, raising renewed interest in whether early intervention might favorably alter the natural history of biologically high-risk disease. In this review, we critically examine the evolution of prognostication in early-stage CLL, integrate contemporary molecular and clinical risk models, and summarize evidence from both historical chemotherapy-era studies and modern early-intervention trials. We discuss key unresolved controversies, including reliance on surrogate endpoints, the risks of overtreatment, and the persistent absence of an overall survival benefit across all early-treatment strategies. Finally, we outline future research priorities, including refined genomic stratification, minimal residual disease-driven (MRD)-driven approaches, and combination targeted therapies currently under investigation. Despite renewed interest in preemptive treatment, available evidence supports continued observation for asymptomatic patients outside clinical trials.

Humans↗

B cell chronic lymphocytic leukaemia/small lymphocytic lymphoma: role of ZAP70 determination on bone marrow biopsy specimens.

BACKGROUND: The course of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) partly depends on the mutational status of the variable region of immunoglobulin heavy chain genes (IgV(H)), which defines two subgroups of tumours: mutated and unmutated. The expression of zeta-associated protein 70 (ZAP70) is significantly associated with the more aggressive unmutated forms. AIMS: To assess the feasibility of the ZAP70 immunohistochemical test on bone-marrow biopsy (BMB) specimens and to compare the results with those of western blotting (WB) and IgV(H) mutational status assessed on neoplastic cells from peripheral blood. METHODS: 26 patients with CLL/SLL detected on BMB and with known IgV(H) mutational status were selected. ZAP70 was determined by immunohistochemistry (IHC) comparing three antibodies from different sources (Upstate, Cell Signaling, Santa Cruz, California, USA) and two different methods (APAAP and EnVision(+)). In 23 cases, ZAP70 WB results were also available. RESULTS: ZAP70 determination on BMB specimens turned out to be easily feasible with routine procedures with reagents from Upstate and Cell Signaling. The results were concordant with those obtained with WB and mutational status analysis in >80% of the cases with both reagents. Three of four discordant cases were mutated/ZAP70 positive, with two staining weakly for ZAP70 on both WB and IHC. CONCLUSIONS: The study confirms the role of ZAP70 as a possible surrogate of mutational status and emphasises its application in routine diagnostics; it discloses a small subset of discordant cases (mutated/ZAP70 weakly positive) that clinically cluster with the more favourable forms.

Aged↗

Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells.

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells.

Animals↗

B lymphocytes in humans express ZAP-70 when activated in vivo.

ZAP-70 is a protein tyrosine kinase initially found in T and NK cells. Recently, this important signaling element was detected in leukemic B cells from a subgroup of patients with B cell chronic lymphocytic leukemia (B-CLL). In this study, ZAP-70 was detected in normal B cells from human tonsils, but not from peripheral blood. The cDNA sequence of B cell ZAP-70 was the same as that in T cells. Germinal center B cells and plasma cells had a substantial proportion of ZAP-70+ cells, while memory and follicular mantle B cells, which contain low numbers of activated B cells, expressed relatively little ZAP-70. A cell fraction of IgD+, CD38+ B cells, which are comprised of many in vivo activated B cells, exhibited the highest levels of ZAP-70. Density gradient fractionation of tonsil B cells confirmed that ZAP-70 was not expressed by resting B cells, but was expressed by buoyant, in vivo activated B cells. In these B cells, the expression of ZAP-70 correlated with that of CD38 and not with that of CD5, a hallmark of B-CLL cells. B-CLL cells are activated cells and their ZAP-70 expression reflects a normal property of activated B cells populations rather than a neoplastic aberration.

ADP-ribosyl Cyclase 1↗

Evolving view of the in-vivo kinetics of chronic lymphocytic leukemia B cells.

B-cell chronic lymphocytic leukemia (B-CLL) has long been considered a disease of "accumulation," due to a presumed defect in programmed cell death. Recent data, however, suggest that B-CLL cells are born at a normal to an accelerated rate, with the rate of proliferation varying among patients. In addition, differences in birth rates, activation state, and inducibility appear to exist among subpopulations of cells within individual leukemic clones. The extent to which such dissimilarities influence clinical course and outcome is still unclear. This review examines the evidence supporting the existence of a proliferative compartment in B-CLL and the role that proliferating cells might play in the progression and evolution of this disease.

Cell Proliferation↗

Therapeutically promising PNA complementary to a regulatory sequence for c-myc: pharmacokinetics in an animal model of human Burkitt's lymphoma.

In Burkitt's lymphoma (BL) cells c-myc is often translocated in proximity to the Emu enhancer of the Ig gene locus. This translocation causes c-myc hyperexpression and an increase in the cells' proliferative capacity. A peptide nucleic acid (PNA) complementary to enhancer Emu intronic sequence (PNAEmu), linked to a nuclear localization signal (NLS), selectively and specifically blocks the expression of the c-myc oncogene under Emu control in vitro, suggesting potential therapeutic use. To explore this issue further, we have determined the pharmacokinetics of (14)C-labeled PNAEmu in SCID mice where a human tumor is established by inoculation of cells from a BL cell line. The data demonstrate that the compound has a relatively long life in vivo in tissues and, in particular, in BL tumor mass. Furthermore, in this animal model, PNAEmu shows low or no toxicity. All these results are in favor of a successful preclinical application in a BL human tumor animal model of a PNA targeting a regulatory, nontranscribed DNA sequence that can selectively inhibit the hyperexpression of a translocated gene linked to neoplastic cell expansion.

Animals↗

In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells.

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.

Aged↗

Immunoglobulin gene mutation patterns and heterogeneity of marginal zone lymphoma.

Marginal zone (MZ) or MZ-like B-cells "home" outside the follicles of peripheral lymphoid tissues. Prototypic examples of these B-cells are those that home the MZ of the spleen, although B-cells with similar phenotypic and functional features can be found in the subepithelial (SE) areas of tonsil, in the Peyer's patches, in the lymph nodes, and in the thymic medulla. MZ-like B-cells also develop in mucosa-associated lymphoid tissue (MALT) and other sites (salivary glands, thyroid) that acquire organized lymphoid tissue after chronic antigenic stimulation, such as Hp infection in gastric maltomas or autoimmune insult in Sjogren's syndrome. Beside the so-called extranodal B-cell MALT lymphoma, the splenic MZ lymphoma with or without circulating villous lymphocytes and monocytoid lymphoma also are thought to originate from MZ B-cells. Normal MZ-like B-cells can be isolated from different tissue sources (tonsil, spleen) by using combination Percoll gradients, magnetic fractionation, and fluorescence-activated cell sorting. Analyses of morphology, phenotype, and functions of these purified MZ B-cells demonstrated that in humans, MZ B-cells are rather heterogeneous, comprising virgin and memory B-cells and of cells specialized in different types of human responses. In this chapter, we review the main procedures used to isolate and test the phenotypic and functional features of the MZ B-cells from human tonsil and spleen in our laboratory and discuss their characteristics by comparing them with the MZ B-cell-derived lymphomas.

B-Lymphocytes↗

Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia.

Previous studies suggest that the diversity of the expressed variable (V) region repertoire of the immunoglobulin (Ig)H chain of B-CLL cells is restricted. Although limited examples of marked constraint in the primary structure of the H and L chain V regions exist, the possibility that this level of restriction is a general principle in this disease has not been accepted. This report describes five sets of patients, mostly with unmutated or minimally mutated IgV genes, with strikingly similar B cell antigen receptors (BCRs) arising from the use of common H and L chain V region gene segments that share CDR3 structural features such as length, amino acid composition, and unique amino acid residues at recombination junctions. Thus, a much more striking degree of structural restriction of the entire BCR and a much higher frequency of receptor sharing exists among patients than appreciated previously. The data imply that either a significant fraction of B-CLL cells was selected by a limited set of antigenic epitopes at some point in their development and/or that they derive from a distinct B cell subpopulation with limited Ig V region diversity. These shared, stereotyped Ig molecules may be valuable probes for antigen identification and important targets for cross-reactive idiotypic therapy.

Amino Acid Sequence↗

Recent advances in the molecular biology and immunobiology of chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) has long been viewed as a relatively homogeneous disease caused by the accumulation of monoclonal immature, immunoincompetent B cells with faulty apoptotic capacities. However, recent evidence, reviewed here, demonstrates that at least two different B-CLL subgroups exist with different clinical courses and outcomes. The malignant cells from both B-CLL subgroups are antigen-experienced cells that have a normal apoptotic apparatus and turnover continually. The leukemic cells of the two B-CLL subgroups have engaged antigen before transformation, although primarily the cells of patients in the poor outcome subgroup can respond to antigens following transformation. The difference in the ability to respond to antigen as a full-fledged B-CLL probably accounts for the different biological features and clinical outcomes of the patients in these subgroups.

Antigens, CD↗

Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia.

Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged V(H)DJ(H) and V(L)J(L) genes of 25 non-IgM-producing B-CLL cases, we found five IgG(+) cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb's reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG(+) B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both.

B-Lymphocytes↗

Inhibition of the translocated c-myc in Burkitt's lymphoma by a PNA complementary to the E mu enhancer.

In Burkitt's Lymphoma there is an up-regulation of the c-myc oncogene caused by its translocation from chromosome 8 to chromosome 14, often close to the E mu enhancer of the immunoglobulin heavy chain locus (IgH). In Burkitt's Lymphoma cells, a peptide nucleic acid complementary for a specific unique E mu intronic sequence selectively blocked the expression of the c-myc oncogene under E mu control but not of other c-myc alleles. This Peptide Nucleic Acid also inhibited IgM expression in B cells. The finding that PNAs specific for a regulatory noncoding sequence can block gene expression has important conceptual and practical implications.

Apoptosis↗

Telomere length and telomerase activity delineate distinctive replicative features of the B-CLL subgroups defined by immunoglobulin V gene mutations.

Patients with B-cell chronic lymphocytic leukemia (B-CLL) segregate into subgroups with very different survival times. Because clinical observations suggest that leukemic cells accumulate at different rates, we measured telomere length and telomerase activity in B-CLL cells to distinguish differences in cellular replication. Our data indicate that the telomeres of B-CLL cells are shorter than telomeres of B cells from healthy subjects, indicating that the leukemic cells have a prolonged proliferative history. Leukemic cells of the immunoglobulin V gene mutation subgroups differ in telomere length and telomerase activity. B lymphocytes from the subgroup with poor outcome and with limited IgV gene mutations have uniformly shorter telomeres and more telomerase activity than those from the subgroup with better outcome and with considerable mutations. Differences in telomere length appear to largely reflect the proliferative histories of precursors of the leukemic cells, although differences in cell division, masked by the action of telomerase, cannot be excluded. These results may provide insight into the stages of maturation and the activation pathways of the cells that give rise to B-CLL. In addition, they reinforce the concept that B-CLL is not simply an accumulative disease of slowly dividing B lymphocytes but possibly one of B cells with extensive proliferative histories.

Antigens, Differentiation, B-Lymphocyte↗

Maintenance of B lymphocyte-related clones in the cerebrospinal fluid of multiple sclerosis patients.

A longitudinal study of Ig V gene segments utilized by B cells from the cerebrospinal fluid (CSF) of two patients with multiple sclerosis (MS) was carried out using RT-PCR methodologies. One patient with a relapsing-remitting (RR)-MS was investigated at onset and at relapse, 1 year later. A patient with secondary-progressive (SP)-MS was tested 9 and 13 years after disease onset. Sequence analyses of V(H)DJ(H) segments bearing V(H)3 and V(H)4 that were obtained from Cgamma cDNA genes demonstrated a substantial proportion of shared clones in the samples taken at different times; these clones were identical or closely related, i.e. had the same third complementary determining region (CDR) of the H chain variable region gene (HCDR3) with different mutations in the V(H) segment. Collectively, these data demonstrate that in MS patients there is a strong selective pressure, which could be exerted by antigen (or autoantigen) stimulation, for the maintenance and partial diversification of certain V(H)DJ(H) Cgamma sequences.

Amino Acid Sequence↗

The human marginal zone B cell.

This study describes the features of the marginal zone (MZ) B cells of human tonsils and spleens and compares them with those of the follicular mantle (FM) B cells from the same tissues. The two B cell subpopulations displayed marked differences in phenotype, in response capacity to T cell-independent antigens and polyclonal B cell activators, and in presentation of antigens to T cells. FM B cells expressed surface CD5, and hence should be considered as B1 cells by current nomenclature. Fractionation of MZ B cells according to the presence or absence of surface IgD revealed the presence of two subsets. These subsets were characterized by different properties, including the presence of Ig V(H) gene mutations and the response capacity to TI-2 antigens, this latter property being associated with IgD-positive cells. Comparison of the data with those reported for mice revealed that human MZ B cells had strong analogies with both the murine MZ and B1 cells. In contrast, human B1 cells (that is, CD5-positive FM cells) were considerably different, an observation that should prompt further studies. Indeed, B cells with characteristics analogous to those of murine B1 cells were detected in small but definite proportions in the peripheral blood and tonsils. If the current distinction into B1 and B2 cells has to be maintained also for humans, it is likely that only these CD5-positive cells rather than the FM B cells should be called B1 cells.

Animals↗

Biology and treatment of chronic lymphocytic leukemia.

Major advances have occurred in our understanding of the biology, immunology, and opportunities for treatment of chronic lymphocytic leukemia (CLL) in recent times. Surface antigen analysis has helped us define classical CLL and differentiate it from variants such as marginal zone leukemia, mantle cell leukemia, and prolymphocytic leukemia. An important observation has been that the B-cells in indolent types of CLL, which do not require therapy, have undergone somatic hypermutation and function as memory B-lymphocytes whereas those more likely to progress have not undergone this process. Section I by Dr. Nicholas Chiorazzi encompasses emerging elements of the new biology of CLL and will address the types of somatic hypermutation that occur in CLL cells and their correlation with other parameters such as telomere length and ZAP70 status. In addition he addresses the concept of which cells are proliferating in CLL and how we can quantitate the proliferative thrust using novel methods. The interaction between these parameters is also explored. Section II by Dr. Thomas Kipps focuses on immune biology and immunotherapy of CLL and discusses new animal models in CLL, which can be exploited to increase understanding of the disease and create new opportunities for testing the interaction of the CLL cells with a variety of elements of the immune system. It is obvious that immunotherapy is emerging as a major therapeutic modality in chronic lymphocytic leukemia. Dr. Kipps addresses the present understanding of the immune status of CLL and the role of passive immunotherapy with monoclonal antibodies such as rituximab, alemtuzumab, and emerging new antibodies. In addition the interaction between the CLL cells and the immune system, which has been exploited in gene therapy with transfection of CLL cells by CD40 ligand, is discussed. In Section III, Dr. Michael Keating examines the question "Do we have the tools to cure CLL?" and focuses on the fact that we now have three distinct modalities, which are able to achieve high quality remissions with polymerase chain reaction (PCR) negativity for the immunoglobulin heavy chain in CLL. These modalities include initial chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab, the use of alemtuzumab for marrow cytoreduction in minimal residual disease and allogeneic bone marrow transplants. The emergence of non-ablative marrow transplants in CLL has led to the broadening of the range of opportunities to treat older patients. The addition of rituximab to the chemotherapy preparative regimens appears to be a significant advance. The combination of our increased understanding of the biology, immune status, and therapy of CLL provides for the first time the opportunity for curative strategies.

Animals↗

Chromosome aberrations evaluated by comparative genomic hybridization in B-cell chronic lymphocytic leukemia: correlation with CD38 expression.

BACKGROUND AND OBJECTIVES: B-cell chronic lymphocytic leukemia (B-CLL) results from the accumulation of monoclonal CD5+ B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous. Clinical studies indicate that CD38+ B-CLL are characterized by a more aggressive clinical course than are CD38- B-CLL. On the basis of these studies and considering the established correlation between specific chromosome aberrations and the clinical course of B-CLL, it is possible that CD38+ B-CLL cases are also characterized by specific subsets of chromosomal alterations. DESIGN AND METHODS: Comparative genomic hybridization (CGH) was performed on purified B-cells from peripheral blood of 52 patients with B-CLL in order to detect chromosome imbalance. The immunophenotype of the patients, including CD38 expression, was also determined by flow cytometry. The results of CGH experiments were then compared with CD38 expression. RESULTS: We found a clear correlation between the presence of chromosomal imbalances and CD38 expression: 13/16 CD38+ cases had chromosome imbalances, most of them (12/13) correlated with a poor prognosis. Among the CD38- B-CLL patients, only 8/36 displayed chromosome imbalances; the only three cases with loss in 13q as a single aberration, considered a good prognostic marker, were in this group. Moreover, we found that cytogenetic alterations were also more complex in the CD38+ B-CLL subset, since 9/10 with two or more aberrations were in the CD38+ group. INTERPRETATION AND CONCLUSIONS: Collectively, the data reinforce the value of CD38 as a prognostic factor and indicate that genotypic/phenotypic features distinguish B-CLL subsets.

ADP-ribosyl Cyclase↗